Unlocking the potential of single-photon wide-field microscopy

释放单光子宽视场显微镜的潜力

• 批准号: EP/Y022491/1
• 负责人: Dirk-Peter Herten
• 金额: $ 80.55万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FY022491%2F1
• 关键词: Unlocking; potential; single; photon; wide

This project aims at transforming wide-field microscopy by adding single photon capabilities that are so far only available in confocal microscopy. We will use the latest generation of single photon sensitive imaging sensors with improved photon detection efficiency and embedded data processing circuits to enable the recording of single photons at MHz rate with picosecond accuracy. This will significantly reduce the readout noise and enable different new imaging modes in wide-field microscopy such as fluorescence lifetime imaging and photon correlation imaging which can be used for multiplexing or molecular counting. In this project, we will focus on improving different super-resolution microscopy techniques for imaging fixed and living cells. For instance, the localisation precision in single-molecule localisation microscopy (SMLM) will benefit not only from the reduced noise levels but also from the possibility to distinguish and reject instantly scattered light from the delayed light emission of the fluorescent labels. Moreover, the embedded photon histogramming capabilities will allow to implement fluorescence lifetime-based multiplexing and thereby increase the number of different labels that can be used in a sample. We will also explore the potential for probing molecular interactions in cells using single-molecule Forster Resonance Energy Transfer. Overall, we expect that this will improve the achievable resolution, add inherent quantitative information to the image data and increase the number of proteins that can be simultaneously imaged. We will explore the application of single-photon SMLM by imaging protein complexes of tetraspanins in the plasma membrane of fixed cells.Furthermore, we want to increase the spatial resolution of super-resolution optical fluctuation imaging (SOFI) and enhance its capabilities in live-cell super-resolution microscopy. Here, the high time resolution of the single photon sensitive imaging sensor will allow us to extend the timescale that can be used for correlating the intensity fluctuations in SOFI. This is very relevant because the majority of optical fluctuations occur on the microsecond timescale which is not covered by current imaging sensors such as EMCCD and sCMOS cameras. Thereby, we expect a significant improvement in the achievable resolution and at the same time a better separation of fluorophores with different properties. We also expect to overcome a major limitation in SOFI by increasing the number of fluorescent labels that can be used in SOFI experiments. Like for single-photon SMLM, we will implement time gating and fluorescence lifetime multiplexing to further reduce noise and increase the number of probes that can be simultaneously imaged. We will explore the suitability of single-photon SOFI by imaging visualising the same protein complexes of tetraspanins and their dynamics in the plasma membrane of living cells.Overall, we expect that the single-photon super-resolution microscopy developed in this project will significantly improve the achievable spatial resolution due to a significantly reduced noise level and rejection of immediate scattering. At the same time, single-photon wide-field microscopy will enable additional imaging modes such as fluorescence lifetime imaging microscopy (FLIM) which will increase the number of simultaneously imaged targets, and photon correlation imaging which will enable quantitative molecular imaging as the first quantum imaging technique in wide-field fluorescence microscopy. The successful development of single-photon super-resolution microscopy will be door opener for other imaging modes such as fluorescence correlation imaging of fast molecular processes. In summary, this will lead to a step change in wide-field microscopy with great prospect to transform the way we can image molecular scale structures and processes in the life sciences and in biomedical research.

该项目旨在通过添加迄今为止仅在共焦显微镜中可用的单光子功能来改变宽视场显微镜。我们将使用最新一代的单光子敏感成像传感器，该传感器具有更高的光子探测效率和嵌入式数据处理电路，能够以兆赫兹速率和皮秒精度记录单光子。这将显着降低读出噪声，并在宽视场显微镜中实现不同的新成像模式，例如可用于多重分析或分子计数的荧光寿命成像和光子相关成像。在这个项目中，我们将重点改进用于固定细胞和活细胞成像的不同超分辨率显微镜技术。例如，单分子定位显微镜（SMLM）的定位精度不仅受益于噪声水平的降低，而且还受益于区分和拒绝来自荧光标记的延迟光发射的即时散射光的可能性。此外，嵌入式光子直方图功能将允许实现基于荧光寿命的多路复用，从而增加可在样品中使用的不同标记的数量。我们还将探索利用单分子福斯特共振能量转移探测细胞内分子相互作用的潜力。总的来说，我们预计这将提高可实现的分辨率，为图像数据添加固有的定量信息，并增加可同时成像的蛋白质数量。我们将通过对固定细胞质膜中四跨膜蛋白的蛋白质复合物进行成像来探索单光子SMLM的应用。此外，我们希望提高超分辨率光学波动成像（SOFI）的空间分辨率并增强其在活体成像中的能力。细胞超分辨率显微镜。在这里，单光子敏感成像传感器的高时间分辨率将使我们能够延长可用于关联 SOFI 中强度波动的时间尺度。这是非常相关的，因为大多数光学波动发生在微秒时间尺度上，而当前的成像传感器（例如 EMCCD 和 sCMOS 相机）无法覆盖这一时间尺度。因此，我们期望可实现的分辨率显着提高，同时更好地分离具有不同特性的荧光团。我们还希望通过增加可用于 SOFI 实验的荧光标记的数量来克服 SOFI 的主要限制。与单光子 SMLM 一样，我们将实施时间选通和荧光寿命复用，以进一步降低噪声并增加可同时成像的探针数量。我们将通过成像可视化相同的四跨膜蛋白复合物及其在活细胞质膜中的动态来探索单光子 SOFI 的适用性。总的来说，我们预计该项目中开发的单光子超分辨率显微镜将显着改善由于显着降低的噪声水平和拒绝即时散射，可实现的空间分辨率。同时，单光子宽视场显微镜将启用额外的成像模式，例如荧光寿命成像显微镜（FLIM），它将增加同时成像目标的数量，以及光子相关成像，这将使定量分子成像成为第一个量子成像技术。宽视场荧光显微镜成像技术。单光子超分辨率显微镜的成功开发将为其他成像模式（例如快速分子过程的荧光相关成像）打开大门。总之，这将导致宽视场显微镜的巨大变革，并有望改变我们在生命科学和生物医学研究中对分子尺度结构和过程进行成像的方式。